Overexpression of the mTOR alpha4 phosphoprotein activates protein phosphatase 2A and increases Stat1alpha binding to PIAS1.

Alpha4 phosphoprotein in the mTOR pathway is a prolactin (PRL)-downregulated gene product that interacts with the catalytic subunit of serine/threonine protein phosphatase 2A (PP2Ac) in rat Nb2 lymphoma cells. Transient overexpression of alpha4 in COS-1 cells inhibited PRL-inducible interferon-regulatory-1 (IRF-1) promoter activity, but the mechanism underlying this inhibition was not known. The present study showed a stable alpha4-PP2Ac complex that was not dissociated by rapamycin in COS-1 cells. Transient overexpression of alpha4 in COS-1 cells had no effect on endogenous PP2Ac protein levels but significantly increased PP2Ac carboxymethylation and PP2A activity as compared to controls. The increased PP2A activity was accompanied by decreased phosphorylation of eukaryotic initiation factor 4E-binding protein (4E-BP1) but had no effect on Stat phosphorylation. However, overexpressed alpha4 decreased arginine methylation of Stat1alpha and increased Stat1alpha binding to the Stat1alpha-specific inhibitor, PIAS1. In summary, ectopic alpha4 increased PP2A activity in COS-1 cells and this was accompanied by Stat1alpha hypomethylation and increased Stat1alpha-PIAS1 association. These events would inhibit Stat action and ultimately inhibit PRL-inducible IRF-1 promoter activity.

Prolactin activates mammalian target-of-rapamycin through phosphatidylinositol 3-kinase and stimulates phosphorylation of p70S6K and 4E-binding protein-1 in lymphoma cells.

Mitogens activate the mammalian target-of-rapamycin (mTOR) pathway through phosphatidylinositol 3-kinase (PI3K). The activated mTOR kinase phosphorylates/ activates ribosomal protein S6 kinase (p70S6K) and phosphorylates/inactivates eukaryotic initiation factor 4E-binding protein-1 (4E-BP1), resulting in the initiation of translation and cell-cycle progression. The prolactin receptor signaling cascade has been implicated in crosstalk with the mTOR pathway, but whether prolactin (PRL) directly activates mTOR is not known. This study showed that PRL stimulated the phosphorylation of mTOR, p70S6K, Akt, and Jak2 kinases in a dose- and time-dependent manner in PRL-dependent rat Nb2 lymphoma cells. PRL-stimulated phosphorylation of mTOR was detected as early as 10 min, closely following the phosphorylation of Akt (upstream of mTOR), but preceding that of the downstream p70S6K. PRL activation of mTOR was inhibited by rapamycin (mTOR inhibitor), LY249002, and wortmannin (P13K inhibitors), but not by AG490 (Jak2 inhibitor), indicating that it was mediated by the P13K/Akt, but not Jak2, pathway. PRL also stimulated phosphorylation of 4E-BP1 in Nb2 cells. PRL-induced phosphorylation of p70S6K and 4E-BP1 was inhibited by rapamycin, but not by okadaic acid (inhibitor of protein phosphatase, PP2A). PRL induced a transient interaction between p70S6K and the catalytic subunit of PP2A (PP2Ac) in 1 and 2 h, whereas a PP2Ac-4E-BP1 complex was constitutively present in quiescent and PRL-treated Nb2 cells. These results suggested that p70S6K and 4E-BP1 were substrates of PP2A and the inhibition of mTOR promoted their dephosphorylation by PP2A. In summary, PRL-stimulated phosphorylation of mTOR is mediated by PI3K. PRL-activated mTOR may phosphorylate p70S6K and 4E-BP1 by restraining PP2A.